Type 1 diabetes mellitus (T1DM) is an auto immune disease characterised by metabolic dysfunction, most notably dysregulation of glucose metabolism, accompanied by characteristic long-term vascular and neurological complications. T1DM is one of the commonest autoimmune diseases, affecting one in 250 individuals in the US where there are approximately 10,000 to 15,000 new cases reported each year, and the incidence is rising. The highest prevalence of T1DM is found in northern Europe, where more than 1 in every 150 Finns develops T1DM by the age of 15. In contrast, T1DM is less common in black and Asian populations where the frequency is less than half that among the white population.
T1DM is characterised by absolute insulin deficiency, making patients dependent on exogenous insulin for survival. Prior to the acute clinical onset of T1DM with symptoms of hyperglycaemia there is a long asymptomatic preclinical period, during which insulin-producing beta cells are progressively destroyed. The autoimmune destruction of beta cells (β cells) is associated with lymphocytic infiltration. In addition, abnormalities in the presentation of MHC Class I antigens on the cell surface have been identified in both animal models and in human T1DM. This immune abnormality may explain why humans become intolerant of self-antigens although it is not clear why only beta cells are preferentially destroyed.
There is a need for new means of treating T1DM, which the substances and methods described herein will address.
There is ample evidence that CD8 cells are involved in the disease process that leads to T1DM. Histological analysis of the islets in an affected individual shows infiltration by CD8 T cells. In animal models of T1DM, the disease process may be transferred from a diseased animal to a healthy animal using CD8 T cells. There is a genetic association between the development of T1DM and certain HLA class I molecules that are critical for CD8 target recognition. Finally, activated CD8 T cells are present in the circulation of high-risk subjects who develop T1DM.
There is an emerging interest in defining the peptide epitopes recognised by CD8 T cells involved in anti-islet autoimmune responses. Identification of epitopes is important for understanding mechanisms of disease development, developing laboratory assays to monitor islet damage and designing therapeutic interventions to halt disease.
The peptide epitopes that form complexes with HLA class I molecules are derived from proteins in the cell cytosol. In the case of an autoimmune disease like T1DM, it can be assumed that the proteins are specific to the cell targeted in the disease. In addition, the epitopes are likely to be from a protein known, from other evidence, to be involved in the autoimmune process as a target (termed an autoantigen). A protein called preproinsulin fits these criteria. It is specific to the β cells destroyed in T1DM. Preproinsulin (PPI) is a precursor protein that gives rise to insulin. Insulin is present in storage granules that occupy most of the β cell cytosol. Insulin is known to be the target of the autoimmune process in T1DM from studies showing the presence of insulin-specific autoantibodies and autoreactive CD4 T cells in most patients who develop the disease. As yet, there are no data on the epitopes of PPI that may be used as targets by CD8 T cells in T1DM.
Previously, methods have been used to try to identify epitopes. At least three approaches have been used previously:
1. Epitope prediction. Most HLA class I molecules have a “preferred” configuration of peptides to which they can bind, termed a “motif”. There are publicly available applications that enable one to search a protein for stretches of sequence that carry the required motif. However, this approach provides no information about whether the epitopes identified are actually generated in vivo. Many peptides have the capability to bind to a class I HLA molecule, so this approach generates many false positives.
2. Generating CD8 T cells. This approach involves cloning CD8 cells that react with a particular protein or peptide. The approach is successful in patients with acute virus infections, where the number of CD8 T cells is high, but is technically much more difficult and demanding in chronic viral infection, autoimmune disease and tumours.
3. Process the protein in vitro. Some of the cellular machinery that packages proteins for HLA display can be manipulated in a cell free environment. It may be possible to incubate PPI with this machinery and then examine the derived peptides for their ability to bind to HLA. This approach has not yet been tested convincingly and requires the additional step of testing the peptides for binding to the HLA.
Chang et al., (2003) Tissue Antigens 62: 408-417 discloses the ALWGPDPAAA peptide as one of 35 synthetic analogues of preproinsulin-derived peptides that are capable of being loaded in-vitro by soluble HLA-A2 molecules. This study also discloses 17 synthetic analogues of preproinsulin-derived peptides that are capable of being loaded in-vitro by soluble HLA-B8 and 17 synthetic analogues of preproinsulin-derived peptides that are capable of being loaded in-vitro by soluble HLA-B15 molecules. This study therefore identifies a large number of candidate peptides, some or all of which might conceivably be loaded by Class I HLAs in vivo. However, it is not possible to identify from this study which, if any, of the disclosed peptides are present in the peptide-MHC complexes used as targets by CD8 T cells in T1DM.
Rathmann et al., (2004) Ann N.Y. Acad Sci 1037: 22-25 discloses that a number of unspecified synthetic preproinsulin-derived peptides, including several peptides from within the signal sequence of preproinsulin are capable of eliciting a T cell response when pulsed on to the surface of PMBCs and CD8+ T cells from two diabetic subjects. However, it is not possible to identify from this publication the identity of the individual peptides to which a T cell response was noted, or to identify the HLA molecule loading these pulsed peptides of the surface of the cells.
To summarise, neither of the above studies either individually or in combination, identify which preproinsulin-derived peptides a native preproinsulin containing cell presents in the context of a given MHC molecule such as HLA-A2. Without such information none of the preproinsulin peptide-HLA complexes disclosed in Chang et al., (2003) Tissue Antigens 62: 408-417 is identifiable as appropriate therapeutic targets.